This proposal is a competitive renewal of program with a long-term objective to elucidate the molecular details of chemotactic signaling in bacteria using chemical biology strategies. A second objective of the proposed research is to ascertain whether our findings in bacteria are relevant for leukocyte chemotaxis. The signaling pathway that results in bacterial chemotaxis serves as a model for understanding two- component signaling in particular and transmembrane signaling in general. Bacteria respond sensitively to changes in chemoeffector concentration over a large concentration range. We hypothesize that bacteria amplify subtle differences in signal concentration through a signaling lattice, and there is mounting evidence for such a model. This model raises several questions: How are proteins organized within the lattice? How does attractant binding alter interactions within the lattice? What is the effect of repellents on the lattice structure? How does covalent modification (phosphorylation and methylation) affect interactions within the lattice? Are there parallels in the signal amplification mechanisms of bacteria and neutrophils? We propose to address these questions using a variety of approaches. These include the synthesis and testing of monovalent and multivalent chemoeffectors, protein footprinting experiments, and x-ray crystallography. Aim 1 is focused on understanding how periplasmic binding proteins (PBPs) function. We have identified a ligand for the periplasmic glucose/galactose binding protein (GGBP), and this ligand inhibits GGBP- mediated chemotaxis. We propose to carry out structural studies to understand how this PBP antagonist functions. We envision that such studies will be useful in devising inhibitors of other bacterial processes mediated by PBPs including biofilm formation and virulence. In aim 2, we plan to continue studies initiated in the past grant period explore the role of inter-chemoreceptor interactions in chemotactic signal amplification in bacteria. In aim 3, we propose to use protein footprinting experiments to investigate protein - protein interactions important in chemotaxis signal transduction. We anticipate that the studies proposed in aims 2 and 3 will provide an increased understanding of how bacteria transduce and amplify signals. In aim 4, we propose to evaluate multivalent ligands as leukocyte chemoattractants. The goal of this aim is to determine whether there are similarities in the mechanisms by which bacteria and leukocytes amplify signals. We anticipate that our studies will facilitate the development of agents that interfere with key two- component signaling pathways;therefore, they may result in the development of new anti-microbial agents. Moreover, neutrophil chemotaxis occurs in the inflammatory/immune response. Thus, conclusions from our studies will be broadly applicable to understanding other signaling pathways involved in human disease.